Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.
3 ABSTRACTSatellite cells (SCs) are muscle-specific stem cells that have a central role in muscle remodeling. Despite their therapeutic potential, SC-based therapies have been met with numerous logistical challenges, limiting their ability to effectively treat systemic muscle diseases, such as Duchenne muscular dystrophy (DMD). Delivery of SC-derived extracellular vesicles (SC-EVs) may unlock the potential offered by SCs and overcome their numerous limitations. Purpose: The purpose of this investigation was to determine the extent to which SC-EVs could restore mitochondrial function in cultured myotubes following oxidative injury. Methods: SC-EVs were isolated from cultured SCs from C57 mice and quantified using nanoparticle tracking analysis (NTA). C2C12 myotubes were cultured and divided into four treatment groups: untreated control, treated for 24 h with SC-EV, 24 h exposure to 50 μ M H 2 O 2 followed by a 24 h recovery period with no treatment, or 24 h exposure to 50 μ M H 2 O 2 followed by a 24 h treatment with SC-EV. Inter-group differences in mitochondrial function were assessed via one-way ANOVA with Tukey post hoc analysis (p<0.05). Results: Given the seeding density used, we calculated that each SC releases approximately 2.35 x 10 5 ± 3.10 x 10 4 EVs per 24 h. Further, using fluorescent microscopy, we verified SC-EVs deliver cargo into myotubes, some of which was localized to the mitochondria. H 2 O 2 exposure resulted in a 42% decline in peak mitochondrial respiration (p=0.0243) as well as a 46% reduction in spare respiratory capacity (p=0.0185) relative to the untreated control group. Subsequent treatment with SC-EVs (3.12x10 8 SC-EV; 24 h) following H 2 O 2 exposure restored 76% of peak mitochondrial respiration (p=0.0187) and 84% of spare respiratory capacity in the damaged myotubes (p=0.0198). SC-EVs did not affect mitochondrial function in the undamaged myotubes. Conclusion: Collectively, these data demonstrate SC-EVs may represent a novel therapeutic approach for treatment of myopathies associated with mitochondrial dysfunction.
Younger age of first exposure (AFE) to American football has been associated with later‐life cognitive deficits in former National Football League players experiencing cognitive and behavioral deficits, but not in other cohorts of high school, college, amateur, or professional contact/collision sports athletes. Although several studies evaluated clinical outcomes, limited information exists among these cohorts and subclinical measures, such as blood biomarkers may serve as an early indicator of future impairments. Extracellular vesicles, including exosome‐like vesicles (ELVs), are released from cells and contain a variety of molecular cargo from the originating cell, and have emerged as reliable biomarker candidates. OBJECTIVE The purpose of this study was to compare plasma ELV concentration, size, and microRNA (miRNA) levels between soccer players reporting AFE to soccer heading before age 10 years (AFE≤10) and soccer players reporting AFE to soccer heading after age 10 years (AFE>10). METHODS College‐aged soccer players self‐reported AFE to soccer heading. Participants were divided into two groups based on their responses: AFE>10 (n=9, 5 males, 12.33±0.24 years) and AFE≤10 (n=18, 11 males, 7.94±0.46 years). Plasma ELV concentration and size were assessed using nanoparticle tracking analysis. Total RNA was isolated from ELVs, followed by performing miRNA first strand cDNA synthesis and subsequently qPCR. Comparisons were made via unpaired t‐test (p<0.05). RESULTS Data are presented as mean ± SE. AFE≤10 was associated with decreased miR‐7844‐5p when compared to AFE>10 (~60%, p=0.03). No changes in miRNA were observed for miR‐92b‐5p, miR‐423‐5p, miR‐24‐3p, miR‐144‐5p, miR‐221‐5p, and miR‐22‐3p. Groups did not differ in concentration (AFE≤10, 8.34 ×109 ELVs/ml plasma ±0.96 ×109 vs AFE>10, 8.39 ×109 EVs/ml plasma ±0.98 ×109) or size (AFE≤10, 117.58 nm ±7.12 vs AFE>10, 106.40 nm ±4.14) of circulating ELVs. CONCLUSION Collectively these data demonstrate that younger AFE to soccer heading was associated with decreased circulating miR‐7844‐5p. Decreased circulating miR‐7844‐5p may be an early indicator of future pathology. Support or Funding Information Supported by NIH‐NINDS R01 NS102157‐01, NIH P20 GM113125, NIH P20 GM103446, NIH R03 HD094594
Muscular pathologies comprise a family of diseases and disorders resulting in the degeneration of skeletal muscle tissue. Current therapeutics are non‐specific to skeletal muscle, which often reduces efficacy and causes harmful off‐target effects in patients. Thus, a myotropic (muscle‐targeted) drug delivery system is needed to shuttle therapeutics to muscle cells to improve efficacy and limit off‐target effects. The objective of this investigation was to develop biological lipid nanoparticles, extracellular vesicles (EVs), with enhanced myotropism. We hypothesized that displaying myotropic transmembrane proteins at the surface of EVs would improve delivery of molecular cargo into muscle cells. To this end, we displayed the myotropic transmembrane proteins MyoMaker (MYMK), MyoMixer (MYMX) and M‐Cadherin (M‐CAD) in the membranes of EVs from Human Embryonic Kidney (HEK293) cells. Using flow cytometry, we measured the incorporation of each protein candidate into the EVs via endogenous green fluorescent protein (GFP) tags included on the cytosolic c‐terminus of each protein. We then measured the delivery of fluorescently‐labeled protein by each myotropic EV candidate in mouse (C2C12) myotubes. In addition to using EVs as delivery vehicles for exogenously loaded therapeutics, the native EV cargo may also be of therapeutic relevance. Given this, we examined the endogenous protein cargo of non‐engineered HEK293‐EVs via mass spectrometry. MYMK displayed the highest degree of incorporation into EVs relative to control HEK293‐EVs (p = 0.006). The MYMK‐EV formulation displayed the greatest increase in protein cargo delivered into myotubes relative to control HEK293‐EVs (~125%, p = 0.0005). Proteomic analysis revealed the top biological processes and molecular functions of HEK293‐EV protein cargo to be ribonucleic acid binding, processing, and metabolism, potentially altering translation in recipient cells. The in vitro data in the present study establish the feasibility of using MYMK‐EVs as a myotropic delivery system. Future investigations are needed to fully elucidate the cell/tissue tropism and physiological effects of MYMK‐EVs in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.